Human blood and lymph contain various types of cells and each cell plays important roles. For example, the erythrocyte carries oxygen; platelets have hemostatic action; and lymphocytes prevent from infection. These various cells originate from hematopoietic stem cells in the bone marrow. Recently, it has been clarified that the hematopoietic stem cells are differentiated to various blood cells, osteoclasts and mast cells by stimulation of various cytokines in vivo and environmental factors. In the cytokines, there have been found, for example, erythropoietin (EPO) for differentiation to erythrocytes; granulocyte colony-stimulating factor (G-CSF) for differentiation to leukocytes; and platelet growth factor (mpl ligand) for differentiation to megakaryocytes which is a platelet producing cells, and the former two have already been clinically applied.
The undifferentiated blood cells are generally classified into two groups consisting of blood precursor cells which are destined to differentiate to specific blood series and hematopoietic stem cells which have differentiation ability to all series and self-replication activity. The blood precursor cells can be identified by various colony assays, however identification method for the hematopoietic stem cells has not been established. In these cells, stem cell factor (SCF), interleukin-3 (IL-3), granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-6 (IL-6), interleukin-1 (IL-1), granulocyte colony stimulating factor (G-CSF) and oncostatin M have been reported to stimulate cell differentiation and proliferation.
Trials for expansion of hematopoietic stem cells in vitro have been examined in order to replace bone marrow transplantation for applying hematopoietic stem cell transplantation therapy or gene therapy. However, when the hematopoietic stem cells are cultured in the presence of the above mentioned cytokines, multi-differentiation activities and self-replication activities, which are originally in the position of the hematopoietic stem cells, gradually disappeared and are changed to the blood cell precursors which are only to differentiate to specific series after 5 weeks of cultivation, and multi-differentiation activity which is one of the specific features of the hematopoietic stem cells, is lost (Wagner et al. Blood 86, 512-523, 1995).
For proliferation of the blood precursor cells, single cytokine is not sufficient to effect, but synergistic action of several cytokines are important. Consequently, in order to proliferate the hematopoietic stem cells in maintaining with specific features of the hematopoietic stem cells, it is necessary to add cytokines which suppress differentiation together with the cytokines which proliferate and differentiate the undifferentiated blood cells. In general, many cytokines, which stimulate proliferation or differentiation of cells, are known, but small numbers of cytokines, which suppressed cell differentiation, are known. For example, leukemia inhibitory factor (LIF) has an action of proliferation of mouse embryonic stem cells without differentiation, but it has no action against the hematopoietic stem cells or blood precursor cells. Transforming growth factor (TGF-xcex2) has suppressive action for proliferation against various cells, but no fixed actions against the hematopoietic stem cells or blood precursor cells.
Not only blood cells but also undifferentiated cells, especially stem cells are thought to be involved in tissue regeneration. These regeneration of tissues and proliferation of undifferentiated cells in each tissue can be applied in various ways by referring to the known reference (Katsutoshi Yoshizato, Regenerationxe2x80x94a mechanism of regeneration, 1996, Yodosha Publ. Co.).
Notch is a receptor type membrane protein, which involves in regulation of nerve cells differentiation found in Drosophila. Homologues of the Notch are found in various animal kinds exceeding to the invertebrate and vertebrate including nematode (Lin-12). Xenopus laevis (Xotch), mouse (Motch) or human (TAN-1).
Ligand of the Notch in Drosophila is known. These are Drosophila Delta (Delta) and Drosophila Serrate (Serrate). Notch ligand homologues are found in various animal kinds as similar to the Notch of receptors (Artavanis-Tsakonas et al., Science 268, 225-232, 1995).
Human Notch homologue, TAN-1 is found widely in the tissues in vivo (Ellisen et al., Cell 66, 649-661, 1991). Three Notch analogous molecules other than TAN-1 are reported (Artavanis-Tsakonas et al., Science 268, 225-232, 1995). Expression of TAN-1 was also observed in CD34 positive cells in blood cells by PCR (Polymerase Chain Reaction) (Milner et al., Blood 83, 2057-2062, 1994). However, in relation to humans, gene and amino acid sequences of human Delta and human Serrate, which are thought to be the Notch ligand, have not been reported as scientific reports in April 1997.
In Drosophila Notch, binding with the ligand was studied and investigated in details, and it was found that the Notch can be bound to the ligand with Ca++ at the binding region, which is a repeated amino acid sequence No. 11 and No. 12 in the amino acid sequence repeat of Epidermal Growth Factor (EGF) like repeating (Fehon et al., Cell 61, 523-534, 1990, Rebay et al., ibid. 67, 687-699, 1991 and International Publication WO 92/19734). EGF-like repeated sequences are conserved in Notch homologues of the other species. Consequently, the same mechanism in binding with ligand is estimated. An amino acid sequence which is called as DSL (Delta-Serrate-Lag-2) near the amino acid terminal, and EGF-like repeated sequence as like in the receptor are conserved in the ligand (Artavanis-Tsakonas et al., Science 268, 225-232, 1995).
EGF-like sequence has been found in thrombomodulin (Jackman et al., Proc. Natl. Acad. Sci. USA 83, 8834-8838, 1986), low density lipoprotein (LDL) receptor (Russell et al., Cell 37, 577-585, 1984), and blood coagulating factor (Furie et al., Cell 53, 505-518, 1988), and is thought to play important roles in extracellular coagulation and adhesion.
Recently, the vertebrate homologues of the cloned Drosophila Delta were found in chicken (C-Delta-1) and Xenopus laevis (X-Delta-1), and it has reported that X-Delta-1 had acted through Xotch in the generation of the protoneuron (Henrique et al., Nature 375, 787-790, 1995 and Chitnis et al., ibid. 375, 761-766, 1995). Vertebrate homologue of Drosophila Serrate was found in rat as rat Jagged (Jagged) (Lindsell et al., Cell 80, 909-917, 1995). According to the Lindsell et al., mRNA of the rat Jagged is detected in the spinal cord of fetal rats. As a result of cocultivation of a myoblast cell line that is forced excess expressed rat Notch with a rat Jagged expression cell line, suppression of differentiation of the myoblast cell line is found. However, the rat Jagged has no action against the myoblast cell line without forced expression of the rat Notch.
A hypothesis has been set up so that Notch and its ligand have an action of differential regulation not only for neuroblasts and myoblasts, but also for various undifferentiated cells, especially blood undifferentiated cells. However, as far as clinical applications in humans, prior known different species such as chicken or Xenopus laevos type Notch ligand have problems with species specificities and antigenicities. Consequently, obtaining prior unknown human Notch ligand is essentially required. The inventor suspected that a molecule having DSL domain and EGF-like domain which are common to Notch ligand molecules and a ligand of the human Notch (TAN-1 etc.), which is a human Delta homologue (hereinafter designates as human Delta) and human Serrate homologue (hereinafter designates as human Serrate), may be found. In addition, these findings may be a candidate for a drug useful for differential regulation of undifferentiated cells.
As a result, in the previous patent application, a gene cloning of three types of molecules including human Delta-1, human Serrate-1 and human Serrate-2 molecules as the human Notch ligand molecules was made, and it was found that these molecules have an action on blood undifferentiated cells. (Refer to WO 97/19172 Differentiation-suppressive polypeptide and WO 98/02458 Differentiation-inhibitor).
As for the human Notch ligand molecule, according to the recent report, partial gene and partial amino acid sequences of the human Delta-1 like molecule, which are, however, incomplete with respect to the specification and disclosure of the full-length sequence, have been-disclosed in the International Publication WO 97/01571. Further, WO 96/27610 discloses total length gene and total length amino acid sequences of human Serrate-1 (humanJagged-1). Also, WO 96/27610 discloses partial length gene and partial length amino acid sequences of human Serate-2 (human Jagged-2). This gene sequence might have erroneous sequences and this gene sequence generates frame shift, which results completely different amino acid sequence of our WO 98/02458, Differentiation-inhibitor. In addition, the said prior arts did not disclose gene cloning of amino terminals. Consequently, the gene sequences and amino acid sequences are incomplete. As a result of searching the gene sequence database, Genebank Release 98 (December 1996), there are four entries about human Serrate-1, i.e. Registered No. HSU61276, HSU3936, HSU77720 and HSU77914, however no other human Notch ligand molecules are found in the said database.
The present invention elucidates the gene sequence and amino acid sequence of novel Notch ligand molecules. Novel Notch ligand molecules and novel therapeutic uses for these molecules are also provided.
In order to search novel human Notch ligands, cross hybridization using the human Delta-1 gene was performed.
To obtain the human Delta-1 gene, methods used in the referential examples 1 and 2, and WO 97/19172 can be applied. Transformed cells, in which a vector PUCDL-1 containing cDNA coding total amino acid sequence of human Delta-1, i.e. DNA containing sequence from No. 179 to No. 2347 in SEQ ID NO: 8, is inserted into E. coli JM109, have been deposited in the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, in Higashi 1-1-3, Tsukuba-shi, Ibaragi-ken, Japan as permanent culture collection E. coli: JM109-pUCDL-1F. Date of deposition was Oct. 28, 1996 as deposition No. FERM BP-5728.
Various lengths of partial genes of this human Delta-1 gene were prepared. Using these partial lengths of genes as probes, numerous cDNA libraries were screened under various hybridization conditions to determine novel Notch ligands by cross hybridization method.
As a result of extensive studies, the isolation of cDNA coding amino acid sequences of novel human Delta-2 have been achieved, a novel molecule having DSL domain common to Notch ligand molecules from human fetal lung cDNA library, and have prepared the expression systems of protein having various forms using the cDNA. Also we have established purification methods of the proteins which were purified and isolated.
Amino acid sequences of novel human Delta-2 are shown in the sequence listings, SEQ ID NO: 1-3. DNA sequence coding these sequences is shown in the sequence listing, SEQ ID NO: 4.
Physiological actions of the these prepared proteins were searched by using many types of cells, for example nerve undifferentiated cells, preadipocytes, hepatocytes, myoblasts, skin undifferentiated cells, blood undifferentiated cells and immune undifferentiated cells. As a result, it has been found that novel human Delta-2 had a differentiation-suppressive action against undifferentiated blood cells, and had a physiological action to maintain an undifferentiated state. Further, it has been found that the molecule has growth suppressive action against vascular endothelial cells.
No significant toxic actions were noted in the toxicity studies on mice, and useful pharmaceutical effects were suggested. Consequently, the pharmaceutical preparations containing the molecule of the present invention, medium containing the molecule of the present invention, and the device immobilized with the molecule of the present invention are novel drugs and medical materials which can maintain the blood undifferentiated cells in the undifferentiated condition. Antibody against human Delta-2 is prepared by using antigen of the said human Delta-2, and purification method of the said antibodies is established. The present invention has completed accordingly.
The present invention relates to a polypeptide comprising at least amino acid sequence of SEQ ID NO: 1 of the sequence listing, a polypeptide comprising at least amino acid sequence of SEQ ID NO: 2 of the sequence listing, and a polypeptide comprising at least amino acid sequence of SEQ ID NO: 3 of the sequence listing. The present invention also relates to the said polypeptides having differentiation suppressive action against undifferentiated cells, the said polypeptides in which the undifferentiated cells are undifferentiated cells except for those of brain and nervous system or muscular system, the said polypeptides in which the undifferentiated cells are undifferentiated blood cells, and the said polypeptides acting on vascular cells. The present invention also relates to a pharmaceutical composition comprising the said polypeptides, and the said pharmaceutical composition having differentiation suppressive action against cells, the said pharmaceutical composition in which the cells are undifferentiated blood cells, and the said pharmaceutical composition having regulatory action against vascular cells. The present invention further relates to a cell culture medium comprising the said polypeptides, the cell culture medium in which the cell is undifferentiated blood cell, and a material having immobilized thereto the polypeptide. Further, the present invention relates to a method for culturing cells using the cell culture medium or the material, and the method in which the cells are undifferentiated blood cells.
The present invention further relates to a DNA coding at least an amino acid sequence of the sequence listing, SEQ ID NO: 1, said DNA coding at least an amino acid sequence of the sequence listing, SEQ ID NO: 2, the DNA coding at least an amino acid sequence of the sequence listing, SEQ ID NO: 3, the DNA having a base sequence from 355 to 927 of the sequence listing, SEQ ID NO: 4, the DNA having base sequence from 355 to 1854 of the sequence listing, SEQ ID NO: 4 and the DNA having base sequence from 355 to 2331 of the sequence listing SEQ ID NO: 4. The present invention still further relates to a recombinant DNA comprising a DNA selected from the group consisting of the DNAs having ligated to a vector DNA which can express said DNA in the host cell, a cell transformed by the recombinant DNA, a method for culturing human cells with the said cells, and a process for production of said polypeptide by culturing the said cells and isolating the compound produced in the cultured mass. The present invention still more further relates to an antibody specifically recognizing a polypeptide having an amino acid sequence of the sequence listing, SEQ ID NO: 3.
Preparation of cDNA necessary for gene manipulation, expression analysis by Northern blotting, screening by hybridization, preparation of recombinant DNA, determination of DNA base sequence and preparation of cDNA library, all of which are series of molecular biological experiments, can be performed according to a description of the conventional textbook for the experiments. The above conventional textbook of the experiments is, for example, Maniatis et al. ed. Molecular Cloning, A laboratory manual, 1989, Eds., Sambrook, J., Fritsch, E. F. and Maniatis, T., Cold Spring Harbor Laboratory Press.
A polypeptide of the present invention has at least polypeptides in the sequence listing SEQ ID NO: 1-3. A mutant and allele, which naturally occur in the nature, are included in the polypeptide of the present invention unless the polypeptides of the sequence listing, SEQ ID NO: 1-3 lose their properties. Modification and substitution of amino acids are described in details in the patent application by the name of Benntt et al. (National Unexam. Publ. WO 96/2645) and can be prepared according to the description thereof.
A DNA sequence coding polypeptides of the sequence listing, SEQ ID NO: 1-3 is shown in the sequence listing, SEQ ID NO: 4, together with their amino acid sequences. In these DNA sequences, even if amino acid level mutation is not generated, naturally isolated chromosomal DNA or cDNA thereof may have a possibility to mutate in the DNA base sequence as a result of degeneracy of genetic code without changing amino acid sequence coded by the DNA. A 5xe2x80x2-untranslated region and 3xe2x80x2-untranslated region do not involve in amino acid sequence determination of the polypeptide, so DNA sequences of these regions are easily mutated. The base sequence obtained by these regeneracies of genetic codes is included in the DNA of the present invention.
Undifferentiated cells in the present invention are defined as cells, which can growth by specific stimulation, and cells, which can be differentiated to the cells having specific functions as a result of the specific stimulation. These include undifferentiated cells of the skin tissues, undifferentiated cells of the brain and nervous systems, undifferentiated cells of the muscular systems and undifferentiated cells of the blood cells. These cells include the cell of self-replication activity which is called as stem cells, and the cell having an ability to generate the cells of these lines. The differentiation-suppressive action means suppressive action for autonomous or heteronomous differentiation of the undifferentiated cells, and is an action for maintaining undifferentiated condition. The brain and nervous undifferentiated cells can be defined as cells having ability to differentiate to the cells of the brain or nerve having specific functions by specific stimulation. The undifferentiated cells of the muscular systems can be defined as cells having ability to differentiate to the muscular cells having specific functions by specific stimulation. The blood undifferentiated cells in the present invention can be defined as cell groups consisting of the blood precursor cells which are differentiated to the specific blood series identified by blood colony assay, and hematopoietic stem cells having differentiation to every series and self-replication activities. Further, in the present invention, vascular cells is defined as general nomination for cells constituting blood vessels, in which vascular endothelial cells is major constituting cells.
The amino acid sequence in the sequence listing, SEQ ID NO: 1 is a sequence of the active center of the novel human Delta-2 of the present invention, from which the signal peptide is deleted, i.e. amino acid sequence from the amino terminal to DSL domain, and corresponds to an amino acid No. 1 to 191 in SEQ ID NO: 3 of the matured full length aminoacid sequence of the novel human Delta-2 of the present invention.
The amino acid sequence in SEQ ID NO: 2 is amino acid sequence of extracellular domain of the novel human Delta-2 of the present invention, from which the signal peptide is deleted, and corresponds to an amino acid No. 1 to 500 in SEQ ID NO: 3 of the matured full length amino acid sequence of the novel human Delta-2 of the present invention.
The amino acid sequence of SEQ ID NO: 3 is the matured full length amino acid sequence of the novel human Delta-2 of the present invention.
The sequence of SEQ ID NO: 4 is cDNA sequence and total amino acid sequence of the novel human Delta-2 of the present invention, which corresponds to the coding region of the said cDNA.
The sequence of SEQ ID NO: 5 is DNA sequence which codes FLAG peptide and amino acid sequence of FLAG peptide used in the present invention.
The sequences of SEQ ID NOs: 6 and 7 are DNA sequences of primers used in referential example 1.
The sequence of SEQ ID NO: 8 is the cDNA sequence and total amino acid sequence of human Delta-1 used in the present invention.
The sequences of SEQ ID NOs: 9, 10, 12 and 13 are DNA sequences of primers used in example 1.
The sequence of SEQ ID NO: 11 is a DNA sequence of a probe used in example 1.
The sequence of SEQ ID NO: 14 is a DNA sequence of a probe used in examples 1 and 2.
The sequences of SEQ ID NOs: 15 to NO: 24 are DNA sequences of primers used in example 3.
The left and right ends of the amino acid sequences in the sequence listings indicate amino terminal (hereinafter designates as N-terminal) and carboxyl terminal (hereinafter designates as C-terminal), respectively, and the left and right ends of the nucleotide sequences are 5xe2x80x2-terminal and 3xe2x80x2-terminal, respectively.
Cloning of unknown human Notch ligand gene can be performed by the following method. During the evolution of the organisms, a part of amino acids sequences and gene sequences of the human Notch ligand is conserved. Cloning can be theoretically possible by using the other Notch legand molecule as a probe. However, in such the cross hybridization, there are many problems, for example, what part is preferable for the probe or how to set up condition for hybridization, and are not so simple. Further, since the cross hybridization process tends to make cloning many numbers of similar genes simultaneously, it takes much times for gene sequence analysis, consequently identification of the objective molecules from the cloned genes is quite difficult.
More than 10 gene fragments have been prepared from the human Delta-1 gene. By using said probes, screenings of cDNA libraries which originate from more than ten different organs were performed under numerous hybridization conditions and washing conditions. A novel Delta like molecule has been sought after.
In the plaque hybridization, clones can be obtained by isotope labeling and non-isotope labeling with the probe. Isotope labeling can be performed by, for example, terminal labeling by using [32P] xcex3-ATP and T4 polynucleotide kinase, or other labeling methods such as nick translation or primer extension method can be applied.
As a result, in example 1, a screening of a human fetal lung cDNA library was prepared using a partial gene of the full length gene of the human Delta-1 as shown in SEQ ID NO: 8 in the sequence listing, i.e. gene sequence shown in SEQ ID NO: 11 in the sequence listing, as a probe. As a result of the first screening, about 120 positive plaques were isolated, and in the second screening, about 80 positive plaques were cloned, then gene sequences of these clones were determined. Most of these cloned genes were the human Delta-1 gene used as a probe. Among them, five clones were found as the novel human Delta-2 gene, which is similar to the human Delta-1 gene, and the objective novel Notch ligand molecule was found.
Among the above five clones, since there were no signal sequence and no amino terminal sequence, a new probe having a gene sequence as shown in SEQ ID NO: 14 was prepared in order to obtain the full length gene. Further, a screening of said human fetal lung cDNA library was repeated with this probe. As a result, cloning of the cDNA coding for the full length of gene was accomplished.
This sequence was compared with the database (Genbank Release 89, December 1996), and found that these were novel sequence.
Examples of plasmids integrated with cDNA are, for example, other than pBluescript KS described in example 1, E. coli originated pBR322, pUC18, pUC19, pUC118 and pUC119 (Takara Shuzo Co. Japan), but the other plasmids can be used, if they can replicate and proliferate in the host cells. Examples of phage vectors integrated with cDNA are, for example, xcexgt10 and xcexgt11, but the other vectors can be used, if they can growth in the host cells. The thus obtained plasmids are transduced into suitable host cells such as genus Escherichia and genus Bacillus using calcium chloride method. Examples of the above genus Escherichia are Escherichia coli K12HB101, MC1061, LE392 and JM109. Example of the above genus Bacillus is Bacillus subtilis M1114. Phage vector can be introduced into the proliferated E. coli by the in vitro packaging method (Enquist and Sternberg, Meth. Enzymol., 68, 281-, 1979).
The said amino acid sequence was analyzed hydrophobic part and hydrophilic part from amino acid sequence according to the method of Kyte-Doolittle (J. Mol. Biol. 151: 105, 1982). As a result, the novel human Delta-2 of the present invention is expressed on cells as cell membrane protein having one transmembrane domain.
According to an analysis of the amino acid sequence of the novel human Delta-2, an amino acid sequence of a precursor of the novel human Delta-2 consists of a 685 amino acid residue shown in the sequence listing, SEQ ID NO: 4, and the signal peptide domain is estimated to correspond to the amino acid sequence of 26 amino acids residue from No. -26 methionine to No. -1 glycine of the sequence listing; extracellular domain: 500 amino acids residue from No. 1 serine to No. 500 serine; transmembrane domain: 26 amino acids residue from No. 501 phenylalanine to No. 526 valine; and intracellular domain: 133 amino acids residue from No. 527 arginine to No. 659 valine. The domain construction is estimated from the amino acid sequences, and an actual presence forms on the cells. Furthermore, a solution may be possible that differs from the above structure, and structural amino acid sequence of each domain hereinabove as defined by possibly changing 5 to 10 amino acids of the sequence.
N-terminal amino acid sequence of the human Delta-2 polypeptide, which is expressed on COS-7 cells, produced and purified as described in example 5, has at least the amino acid sequence started from No. 1 serine in the sequence listing, SEQ ID NO: 2. Similarly, identical N-terminal can be expected, if the said peptide is expressed in the other animal cells.
According to a comparison in the full length amino acid sequence of the novel human Delta-2 of the present invention with other Notch ligand molecules, which has been reported by April 1997, the homology with human Delta-1 (amino acid sequence of SEQ ID NO: 8 in the sequence listing) as a molecule originated from human is 48.5%; with human Serrate-1 (Genbank HSU61276 and HSU73939) is 40.3%; and with human Serrate-2 (Japanese Patent Appln. No. 8-18622, Differentiation-suppressive polypeptide in the name of the present inventors) is 42.7%. The homologies with Delta of other vertebrates are: mouse Delta-1 (D111, Genbank MMDELTA1) 48.7%; flog Delta-1 (Genbank XELXDEL) 47.0%; flog Delta-2 (Genbank XLU70843) 49.7%, and chicken Delta-1 (Genbank GGU26590) 47.9%.
As the result, the human Delta-2 of the present invention is a novel molecule, which has never been reported in humans or in other biological homologues, and is a novel substance having an amino acid sequence different from these substances, and is a novel substance which has been elucidated for the first time by the present inventor. Moreover, no polypeptide having the same amino acid sequence as the novel human Delta-2 has been found by a homology search in other organisms.
The homologues of Notch ligand have an evolutionary conserved common sequence, i.e. a DSL sequence and repeated EGF-like sequence. As a result of a comparison with the novel human Delta-2 and human Delta-1, these conserved amino acid sequences of the novel human Delta-2 are estimated.
Namely, DSL sequence corresponds to 43 amino acids residue from No. 149 cysteine to No. 191 cysteine of the amino acid sequence in the sequence listing, SEQ ID NO: 4. EGF-like sequence exists with 8 repeats wherein, in the amino acid sequence in the sequence listing, SEQ ID NO: 4, the first EGF-like sequence corresponds to the sequence from No. 196 cysteine to No. 224 cysteine; the second EGF-like sequence corresponds to the sequence from No. 227 cysteine to No. 255 cysteine; the third EGF-like sequence corresponds to the sequence from No. 262 cysteine to No. 295 cysteine; the fourth EGF-like sequence corresponds to the sequence from No. 302 cysteine to No. 333 cysteine; the fifth EGF-like sequence corresponds to the sequence from No. 340 cysteine to No. 373 cysteine; the sixth EGF-like sequence corresponds to the sequence from No. 380 cysteine to No. 411 cysteine; the seventh EGF-like sequence corresponds to the sequence from No. 418 cysteine to No. 449 cysteine; and the eighth EGF-like sequence corresponds to the sequence from No. 458 cysteine to No. 491 cysteine.
A part of sugar chain attached is estimated from amino acid sequence of the novel human Delta-2 may be No. 82, 157, 179 and 367 asparagine residue in the sequence listing, SEQ ID NO: 4 as a possible binding site of N-glycoside bonding for N-acetyl-D-glucosamine. 0-glycoside bond of N-acetyl-D-galactosamine is estimated to be a serine or threonine residue rich part. Protein bound with sugar chain is generally thought to be stable in vivo and to have strong physiological activity. Consequently, in the amino acid sequence of polypeptide having sequence of the sequence listing SEQ ID NO: 1, 2 or 3, polypeptides having N-glucoside or 0-glucoside bond with sugar chain of N-acetyl-D-glucosamine or N-acetyl-D-galactosamine is included in the present invention. As shown in example 5, if the human Delta-2 of the present invention is expressed by gene inserted COS-7 cell, at least more than two forms are expressed due to attached sugar chain as the proteins having different molecular weight.
As a result of studies on binding of Drosophila Notch and its ligand, amino acid region necessary for binding with ligand of Drosophila Notch with the Notch is from N-terminal to DSL sequence of the matured protein, in which signal peptide is removed (International Publication WO 92/19734). Further, similarly, studies using Nematode by Fitzgerald and Greenwald (Development, 121, 4275-4282, 1995) clearly indicate that Notch ligand like molecule APX-1 required for Notch like receptor activation is sufficient from amino terminal to DSL domain in the full length sequence.
This fact indicates that a domain necessary for expression of ligand action of human Notch ligand molecule is at least the DSL domain, i.e. a domain containing amino acid sequence from No. 149 cycteine to No. 191, cycteine in the sequence listing, SEQ ID NO: 1, and a domain at least necessary for expression of ligand action of human Delta-2 is novel amino acid sequence shown in the sequence listing, SEQ ID NO: 1, further a domain at least necessary for expression of ligand action of human Delta-2 is novel amino acid sequence shown in the sequence listing, SEQ ID NO: 2.
As shown in example 2, mRNA of the human Delta-2 can be detected by using DNA coding a part or full of gene sequence in the sequence listing, SEQ ID NO: 4. For example, a method for detection of expression of these genes can be achieved by applying with hybridization or PCR by using complementary nucleic acids of above 12 mer or above 16 mer, preferably above 20 mer having nucleic acid sequence of a part of sequence in the sequence listing, SEQ ID NO: 4, i.e. antisense DNA or antisense RNA, its methylated, methylphosphated, deaminated or thiophosphated derivatives. Using the same method, detection of homologues of the gene of other organisms such as mice or gene cloning can be achieved. Further cloning of genes in the genome including humans can be made. Using these genes cloned by such like methods, further detailed functions of the human Delta-2 can be clarified. For example, using the recent gene manipulation techniques, every methods including transgenic mouse, gene targeting mouse or double knockout mouse in which genes relating to the gene of the present invention are inactivated, can be applied. If abnormalities in the genome of the present gene are found, application to gene diagnosis and gene therapy can be made.
A transformant in which vector PBSDL-2, which contains cDNA coding total amino acid sequence of the novel human Delta-2 of the present invention, is transformed into E. coli JM109, has been deposited in the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, of 1-1-3, Higashi, Tsukuba-shi, Ibaragi-ken, Japan, as E. coli: JM109-pBSDL-2. Date of deposit was May 9, 1997, and deposition No. is FBRM BP-5941.
Process for production of the novel human Delta-2 polypeptide can be performed, as shown in example 3, by using expression vector pcDNA 3. Production and purification of various forms of the novel human Delta-2 polypeptide using cDNA, which codes amino acid sequence of the novel human Delta-2 isolated by the above method are known in the references (Kriegler, Gene Transfer and Expression-A Laboratory Manual, Stockton Press, 1990 and Yokota et al. Biomanual Series 4, Gene Transfer and Expression and Analysis, Yodosha Co., 1994). A cDNA coding the amino acid sequence of the isolated said human Delta-2 is ligated to preferable expression vector and it is produced in the host cells of eukaryotic cells such as animal cells and insect cells or prokaryotic cells such as bacteria.
In the expression of the novel human Delta-2 of the present invention, DNA coding polypeptide of the present invention may have the translation initiation codon in 5xe2x80x2-terminal and translation termination codon in 3xe2x80x2-terminal. These translation initiation codon and translation termination codon can be added by using preferable synthetic DNA adapter. Further for expression of the said DNA, promoter is ligated in the upstream of the DNA sequence. Examples of vector are plasmid originated from the above E. coli, plasmid originated from Bacillus, plasmid originated from yeast or bacteriophage such as xcex-phage and animal viruses such as retrovirus and vaccinia virus.
Examples of promoters used in the present invention are any promoters preferable for corresponding to the host cells used in gene expression.
In case that the host cell in the transformation is genus Escherichia, tac-promoter, trp-promoter and lac-promoter are preferable, and in case of host of genus Bacillus, SP01 promoter and SP02 promoter are preferable, and in case of host of yeast, PGK promoter, GAP promoter and ADH promoter are preferable.
In case that the host cell is animal cell, a promoter originated from SV40, promoter of retrovirus, metallothionein promoter and heat shock promoter can be applied.
Expression of the polypeptide of the present invention can be made by using only DNA coding the amino acid sequence of the sequence listing, SEQ ID NO: 1, 2 or 3. However, the protein added with specific function can be produced by using DNA, to which added cDNA coding the known antigen epitope for easier detection of the produced polypeptide or added cDNA coding the immunoglobulin Fc for forming multimer.
As shown in Example 3, we have prepared expression vectors, which express extracellular proteins of the novel human Delta-2, as follow:
1) DNA coding the amino acids from No. 1 to 500 in amino acid sequence in the sequence listing, SEQ ID NO: 2;
2) DNA coding chimera protein, to which added polypeptide having 8 amino acids, i.e. an amino acid sequence consisting of Asp Tyr Lys Asp Asp Asp Asp Lys (hereinafter designates FLAG sequence, an example of DNA sequence coding the same is shown in the sequence listing, SEQ ID NO: 5), in the C-terminal of the amino acids from No. 1 to 500 in amino acid sequence in the sequence listing, SEQ ID NO: 2; and
3) DNA coding chimera protein, to which added Fc sequence from the hinge region of human IgG1 in the C-terminal of the amino acids from No. 1 to 500 in amino acid sequence in the sequence listing, SEQ ID NO: 2, is ligated to each separately with the expression vector pcDNA 3 (INVITROGEN Corp., U.S.A.), then the expression vector expressing the extracellular region of the novel human Delta-2 is prepared.
The expression vector for expression of full length of the novel human Delta-2 can be prepared as follows:
4) DNA coding amino acids from No. 1 to 659 in the sequence listing, SEQ ID NO: 3 and
5) DNA coding chimera protein, to which added polypeptide having FLAG sequence in the C-terminal of amino acids from No. 1 to 659 in the sequence listing, SEQ ID NO: 3 are ligated individually with the expression vector pcDNA to prepare the expression vector, which can express full length of the novel human Delta-2.
The transformants are prepared by using these expression plasmids containing DNA coding the thus constructed said human Delta-2.
Examples of the host are genus Escherichia, genus Bacillus, yeast and animal cells. Examples of animal cells are simian cell COS-7 and Vero, Chinese hamster cell CHO and silk worm cell SF9.
As shown in Example 4, the expression vectors of the above 1)-5) are transduced individually; the novel human Delta-2 is expressed in COS-7 cell (obtainable from the Institute of Physical and Chemical Research, Cell Development Bank, RCB0539), and the transformants, which are transformed by these expression plasmids, can be obtained. Further, the novel human Delta-2 polypeptide can be produced by culturing the transformants under preferable culture condition in medium by known culture method.
As shown in Example 5, the novel human Delta-2 polypeptide can be isolated and purified from the above cultured mass, in general, by the following methods.
For extraction of the substance from cultured microbial cells or cells, microbial cells or cells are collected by known methods such as centrifugation after the cultivation, suspended in preferable buffer solution, disrupted the microbial cells or cells by means of ultrasonication, lysozyme and/or freeze-thawing and collected crude extract by centrifugation or filtration. The buffer solution may contain protein-denaturing agents such as urea and guanidine hydrochloride or surface active agents such as Triton-X. In case of secretion in the cultured solution, the cultured mass is separated by the known method such as centrifugation to separate from microbial cells or cells and the supernatant solution is collected.
The thus obtained novel human Delta-2, which is contained in the cell extracts or cell supernatants, can be purified by known protein purification methods. During the purification process, for confirmation of existence of the protein, in case of the fused proteins such as the above FLAG and human IgGFc, they can be detected by immunoassay using antibody against known antigen epitope and can be purified. In case of not to express as such the fused protein, the antibody in Example 6 can be used for detection.
Antibodies, which specifically recognize human Delta-2, can be prepared as shown in Example 6. Antibodies can be prepared by the methods described in the reference (Antibodies a laboratory manual, E. Harlow et al., Cold Spring Harbor Laboratory) or recombinant antibodies expressed in cells by using immunoglobulin genes isolated by gene cloning method. The thus prepared antibodies can be used for purification of the novel human Delta-2. The human Delta-2 can be detected and assayed by using antibodies which recognize specifically novel human Delta-2 as shown in Example 6, and can be used for diagnostic agents for diseases accompanied with abnormal differentiation of cells such as malignant tumors.
More useful purification method is the affinity chromatography using antibody. Antibodies used in this case are antibodies described in Example 6. For fused protein, antibodies against FLAG in the case of FLAG, and protein G or protein A in the case of human IgGFc as shown in Example 5.
Physiological functions of the thus purified human Delta-2 protein can be identified by various assay methods, for example, physiological activity assaying methods using cell lines and animals such as mice and rats, assay methods of intracellular signal transduction based on molecular biological means, binding with Notch receptor etc.
Actions for blood undifferentiated cells have been observed by using IgG1 chimera protein of novel human Delta-2. As a result, it has been found that, as shown in example 7, in undifferentiated umbilical cord derived blood cells, in which the CD34 positive cell fraction is concentrated, the novel human Delta-2 has suppressive action of colony forming action against blood undifferentiated cells, which show colony formation in the presence of cytokines.
Further as shown in example 8, it has been found that by evaluating LTC-IC (Long-term Culture-initiating Cells), which are positioned from most undifferentiated blood stem cells in the human blood undifferentiated cells, after culturing the undifferentiated umbilical cord derived blood cells, in which the CD34 positive cell fraction is concentrated, in the presence of the human Delta-2 with various cytokines in serum-free medium, the human Delta-2 has an activity to maintain a number of LTC-IC. Further, example 9 shows that the human Delta-2 is Bound with human blood undifferentiated cells.
The results indicate that the human Delta-2 suppresses differentiation of blood undifferentiated cells, and this action is obviously effective for cells from blood stem cells to colony forming cells. These physiological actions are essential for in vitro proliferation of blood undifferentiated cells. Especially, cells cultured in the medium containing human Delta-2 are efficient in recovery of suppression of bone marrow after administration of antitumor agents, accordingly in vitro expansion of hemopoietic stem cells may be possible if other conditions would be completed. Further pharmaceuticals containing the polypeptide of the present invention have protective and reduced actions against the bone marrow suppressive action due to adverse effects of antitumor agents.
In these experiments, the LTC-IC maintaining activity and binding action for blood cells of the novel human Delta-2 of the present invention are stronger than those of the human Delta-1 (WO 97/19172), which has same action as shown by the present inventors.
As shown in example 9, IgG1 chimera protein of the human Delta-2 is bound with CD34 positive blood undifferentiated cells. By this binding activity, the polypeptide of the present invention can be used for isolation and detection of cells. Although the isolation method can be performed by a method using flow cytometer as described in example 9, a method using materials, to which polypeptide of the present invention is immobilized, as described in example 11 may be more convenient. Consequently, cell isolation method using polypeptide of the present invention is included in the present invention. Further, cell isolation method using a material, to which polypeptide of the present invention is immobilized, and device for cell isolation applied with the said isolation method is also included in the present invention. Any cell isolation method using antibodies described in the references is applicable to these isolation devices and isolation methods. For example, Dynabeads of Dynal Corp., Norway, which is a method using combination of magnetic beads and antibodies, can be used.
Further, as shown in example 12, IgG1 chimera protein of the novel human Delta-2 of the present invention has suppressive action against proliferation of intravascular endothelial cells and has inhibitory action against vascularization. Consequently, the polypeptide of the present invention can be used as therapeutic agents for diseases and disease states, which may be cured by suppressing vascularization as proposed by Folkman and Klagsbrun (Science 235, 442-447, 1987). Concrete examples of use are described in the above reference, and are, for example, diseases including malignant tumors.
Suppressive action for differentiation of cells in the undifferentiated cells other than blood cells is expected and stimulating action for tissue regeneration can be expected.
In the pharmaceutical use, polypeptides of the present invention are lyophilized with adding preferable stabilizing agents such as human serum albumin, and are used in dissolved or suspended condition with distilled water for injection when it is in use. For example, preparation for injection or infusion at the concentration of 0.1-1000 xcexcg/ml may be provided. A mixture of the compound of the present invention 1 mg/ml and human serum albumin 1 mg/ml divided in a vial could maintain activity of the said compound for long term. For culturing and activating cells in vitro, lyophilized preparation or liquid preparation of the polypeptide of the present invention are prepared and are added to the medium or immobilized in the vessel for culture. Toxicity of the polypeptide of the present invention was tested. Any polypeptide, 10 mg/kg was administered intraperitoneally in mice, but no death of mice was observed.
In vitro physiological activity of the polypeptide of the present invention can be evaluated by administering to disease model mice or its resembled disease rats or monkeys, and examining recovery of physical and physiological functions and abnormal findings. For example, in case of searching abnormality in relation to hemopoietic cells, bone marrow suppressive model mice are prepared by administering 5-FU series of antitumor agents, and bone marrow cell counts, peripheral blood cell counts and physiological functions are examined in the administered group or the non administered group of mice. Further, in case of searching in vitro cultivation and growth of hemopoietic undifferentiated cells including hemopoietic stem cells, the bone marrow cells of mice are cultured in the groups with or without addition of the compound of the present invention, and the cultured cells are transferred into the lethal dose irradiated mice. Result of recovery is observed with the indications of survival rate and variation of blood counts. These results can be extrapolated to the humans, and accordingly useful effective data for evaluation of the pharmacological activities of the compound of the present invention can be obtained.
Applications of the compound of the present invention for pharmaceuticals include diseases with abnormal differentiation of cells, for example leukemia and malignant tumors. These are cell therapy, which is performed by culturing human derived cells in vitro with maintaining their original functions or adding new functions, and a therapy, which is performed by regenerating without damaged the functions of the originally existed in the tissues by administering the compound of the present invention under the regeneration after tissue injury. Amount of administration may differ in the type of preparation and is ranged from 10 xcexcg/kg to 10 mg/kg.
Further strong physiological activity can be achieved by expression of forming multimer of the polypeptide of the present invention.
Human Delta-2 having multimer structure can be produced by the method of expressing chimera protein with human IgG Fc region as described in the examples 3 and 4 and expressing the multimer having disulfide bond with hinge region of the antibody, or a method expressing chimera protein, in which antibody recognition region is expressed in the C-terminal or N-terminal, and reacting with the polypeptide containing extracellular part of the thus expressed said human Delta-2 and the antibody which recognize specifically the antibody recognition region in the C-terminal or N-terminal. In the other methods, a method, in which a fused protein with only the hinge region of the antibody is expressed and the dimer is formed by disulfide bond, can be mentioned. The multimer of human Delta-2 having higher specific activity than the dimer can be obtained. The said multimer is constructed by fused protein which is prepared for expressing the peptide in the C-terminal, N-terminal or other region. The protein is prepared in the form of forming disulfide bond without effecting in any activities of the other human Delta-2. The multimer structure can also be expressed by arranging one or more peptide, which is selected from polypeptides containing amino acids sequence of the sequence listing, SEQ ID NO: 1, 2 and 3, with genetic engineering method in series or in parallel. Other known methods for providing multimer structure having dimer or more can be applied. Accordingly, the present invention includes any polypeptides containing amino acid sequences described in the sequence listing, SEQ ID NO: 1, 2 and 3 in the form of dimer or more structure prepared by genetic engineering technique.
Further in the other method, multimerization method using chemical cross-linker can be mentioned. For example, dimethylsuberimidate dihydrochloride for cross-linking lysine residue, N-(xcex3-maleimidebutyryloxy) succinimide for cross-linking thiol group of cysteine residue and glutaraldehyde for cross-linking between amino groups can be mentioned. The multimer with dimer or more can be synthesized by applying these cross-linking reactions. Accordingly, the present invention includes any polypeptides containing amino acid sequences described in the sequence listing, SEQ ID NO: 1, 2 or 3 in the form of dimer or multimer structure prepared by chemical cross-linking agents.
In application of medical care in which cells are proliferated and activated in vitro and are returned to the body, human Delta-2 of the form hereinabove can be added directly in the medium, but immobilization can also be made. Immobilization method includes applying amino group or carboxyl group in the peptide, using suitable spacers or the above mentioned cross-linkers, and the polypeptide can be covalently bound to the culture vessels. In example 11, method for preparation of the immobilized material and their effect are illustrated. Accordingly, the present invention includes any polypeptides containing amino acid sequences described in the sequence listing, SEQ ID NO: 1, 2 or 3 in the form of existing on the solid surface.
Since the natural human Delta-2 is cell membrane proteins, differentiation suppressive action in example 7, 8 and 12 can be expressed by cocultivating with cells expressing these molecules and blood undifferentiated cells. Consequently, this invention includes a method for cocultivation of undiferentiated cells with transformed cells by using DNA coding amino acid sequence in the sequence listing, SEQ ID NO: 1, 2 or 3. An example is illustrated in example 10. Expressed cell may be simian COS-7 cell or mouse Balb 3T3 cells as shown in examples, but cells of human origin are preferable, and further expressed cells may be any of human in vivo blood cells and somatic cells rather than cell lines. Consequently, the polypeptide can be expressed in vivo by integrated into vectors for gene therapy. Examples of vectors for gene therapy are retrovirus vector, adenovirus vector or adeno-related virus vector.
This fact suggests that inhibition of binding of the polypeptide having amino acid sequence in the sequence listing, SEQ ID NO: 1, 2 or 3 to these receptors can be used for finding out molecules and compounds for stimulating cell differentiation. The methods include binding experiment using radio isotope, luciferase assay using transcriptional control factors, a down stream molecule of the Notch receptor, and simulation on the computer by X-ray structural analysis. Accordingly, the present invention includes a screening method for pharmaceuticals using polypeptide in the sequence listing, SEQ ID NO: 1, 2 or 3.